Project Summary Alzheimer?s disease (AD) continues to be a major health issue in the US. More than 5.5. million people are currently diagnosed, and this number is expected to triple by 2025 if treatments directed to prevent, halt or re- vert the pathological process are not developed. Thus, it is imperative to better understand molecular pro- cesses that influence brain function and health. Recently, several studies suggest that dysbiosis may modu- late the inflammation associated with AD pathobiology. However, the relationship between dysbiosis and dis- ease state is still poorly understood. The current hypothesis establish that bioactive molecules are transported from the gut (or other colonized tissues) to the brain through the enteric nervous system or promote activation of neuroinflammatory pathways. Studies directed to determine the association between changes in microbiota and AD are based on characterizing fecal bacteria, using 16S ribosomal RNA. This approach presents limita- tions such as lack of consideration of other colonized areas (i.e. mouth, skin, nose), and lack of studies on specific bacterial biomolecules that may be associated with disease state. As far as we know, there is no evi- dence of the establishment of a brain microbiota that directly influence the central nervous system, explaining how dysbiosis could lead to neurodegeneration. We argued that an experimental approach to identify bacterial biomolecules in brain tissue is needed. The proposed research work is directed to uncover the presence of specific bacterial species in normal aging and AD brain and to enhance the understanding between dysbiosis and neurodegeneration. To achieve these goals, we developed an unbiased proteomics ap- proach and stringent datamining pipeline to identify bacterial proteins in normal aging and AD brain tissue from two different brain banks. After filtered bacterial proteins with human orthologues, our preliminary studies demonstrate the detection of bacterial proteins corresponding to specific bacteria species in human frontal cor- tex that differentiate between normal aging and AD. Thus, we are well positioned to test the hypothesis that dysbiosis of brain microbiota plays a role in neurodegeneration. To test this hypothesis, we propose to 1) to study changes in bacterial proteins and bacterial species at different neuropathological stages and 2) to functionally determine the transmission of specific bacteria from the periphery to the brain. The main goal of this project is to demonstrate that changes in brain microbiota has an effect on health and dis- ease. This information will contribute to understanding the mechanisms underlying the role of dysbiosis in neu- rodegeneration and will set the basis for a study of restoration and prevention.